1. Field of the Invention
This invention relates to a fluorescence detection apparatus for detecting a fluorescence signal from a specific substance contained in a sample and quantifying the specific substance from the detected fluorescence signal amount and in particular to a fluorescence detection apparatus useful for monitoring a large number of samples in real time (tracing change of the fluorescence signal amount with time) in a clinical diagnosis field requiring incubation at a predetermined temperature, such as an enzyme reaction.
2. Description of the Related Art
To monitor producing a fluorescent reaction product by an enzyme reaction in real time, etc., it is necessary to detect fluorescence while incubating a sample (reaction liquid) at a predetermined temperature. Moreover, a large number of samples need also to be treated promptly at the same time in fields of clinical diagnosis, etc.
A first method used in the conventional clinical diagnosis field, etc., is a method of detecting fluorescence in order while transporting samples along a temperature-adjusted guide. For example, the temperature of a guide manufactured with a material having good thermal conductivity such as an aluminum alloy is adjusted by a heater, etc., samples placed in a holder are transported along the guide using a chain, a turn table, or the like one or more than one at a time, and a fluorescence signal is detected in order by a fluorescence detector placed along the guide.
In addition, a second method of detecting fluorescence at the same time about a large number of samples, for example, by placing a joint-type sample vessel, titer plate, etc., capable of storing a large number of samples on temperature adjustment means is also known. A fluorescence detection apparatus used for the purpose comprises (a) a plurality of photosensors or (b) a multichannel-type photosensor or has (c) mechanical move means for moving photosensor or light guide (means for guiding a fluorescence signal emitted from a sample vessel to photosensor, such as an optical fiber).
The apparatus (a) is a fluorescence detection apparatus for using as many photosensors as samples for detecting fluorescence at the same time to detect a fluorescence signal separately from each sample. In such an apparatus, it is common practice to use a light guide for dividing excitation light from a light source and guiding to each sample.
The apparatus (b) is a fluorescence detection apparatus for using an image sensor such as CCD or a photodiode array in place of a plurality of photosensors, thereby detecting fluorescence signals from aligned samples as an image in a state in which the positional relationship between light emission points is held. In such an apparatus, it is also common practice to guide excitation light from a light source to each sample by using a division-type light guide, such as an optical device or an optical fiber.
In the apparatus (c), the photosensor is moved mechanically on a large number of samples or samples are moved to the fluorescence detection position of the photosensor in order; most used is a configuration of moving the light guide mechanically. In this configuration, an excitation light guide and a fluorescence light guide are used and the ends of both guides placed on the sample side are made integral with each other, then both guides are moved at the same time, whereby fluorescence is detected while a large number of samples are excited in order.
To use the fluorescence detection apparatus in the conventional arts to monitor change of a fluorescence signal with time from a specific substance contained in a sample in real time while incubating the sample at a predetermined temperature, the following problems are involved:
The first method described above involves the risk of insufficient temperature adjustment accuracy, the limit of the number of treated samples, carry-over, etc., because samples are transported along the temperature-adjusted guide and fluorescence is detected in order. That is, it is difficult to adjust the whole sample transport guide at a uniform temperature and hold the thermal conductivity between the transport guide and each sample constant over the whole guide; consequently, temperature change of the sample may occur during transporting or the samples may differ in temperature. Since fluorescence is detected about the transported samples one at a time, the same sample must be transported repeatedly to monitor change of a fluorescence signal with time for a long time, thus the number of samples that can be treated is limited. Further, the risk of contamination (carry-over) between the samples caused by a sample splash cannot be excluded.
The second method described above can solve the problems of the first method, but may introduce the following new problems:
First, the conventional apparatus (a) comprises a plurality of photosensors, thus the manufacturing costs are increased and the space matching the number of photosensors also becomes necessary. If an attempt is made to miniaturize the apparatus, several photosensors can only be installed because of the limit of the space; after all, the number of samples that can be treated at the same time is only a few. Although use of small-sized photosensors such as photodiodes can also be considered, there is a problem of insufficient sensitivity to feeble fluorescence, and it becomes necessary to correct the sensitivity of each photodiode. Further, the strength of a fluorescence signal is proportional to the excitation light strength and thus if excitation light from the light source is divided, detection sensitivity is worsened; this is also a problem.
Next, the apparatus (b) has insufficient sensitivity to feeble fluorescence and thus is not adequate. To augment insufficient sensitivity, an element for amplifying the light quantity via electron amplification by a microchannel plate (so-called image intensifier) or the like may be used in combination, but is used only in special research application under the present circumstances because of an extremely rise in costs. Since fluorescence from a wide range is detected as an image, there are also problems of unevenness of light quantity detection caused by lens aberration and data processing load caused by an enormous data amount.
With the apparatus (c), the move range is limited because of the limit of the bendability of the light guide and moreover there is a possibility of breaking light guide. Since light communication efficiency is changed because the light guide is bent, it is difficult to make fluorescence detection good in reproducibility. On the other hand, a mechanical move of the photosensor also involves a move of attached cables, etc., thus the move range is limited and there is a possibility of breaking the cable, etc.
In addition, scanner-type fluorescence detection apparatus as disclosed in Japanese patent Unexamined Publication No. 2000-088752 (P2000-88752A) and an apparatus as shown in FIG. 6 invented as means for solving problems of the above-described apparatus are also available. The scanner-type fluorescence detection apparatus described in Japanese patent Unexamined Publication No. 2000-088752 is as follows: As shown in FIGS. 5A AND 5B, sample vessels are arranged like a circular arc and a ring section of a ring-type light guide 21 is placed closely facing the sample vessels with putting a partition plate 23 therebetween and excitation light optical means 25 and fluorescence optical means 26 are fixed to the partition plate for rotation integrally, whereby separately gathered fluorescence signals are communicated through the ring-type light guide 21 to a photosensor 22. In the scanner-type fluorescence detection apparatus as shown in FIG. 6A and 6B, sample vessels are arranged like a plurality of circular arcs and a ring-type light guide 31 is placed facing the sample vessels with a partition plate 33 between and excitation light optical means 35 and fluorescence optical means 36 containing at least one light guide are fixed to the partition plate 33 for rotation integrally, whereby separately gathered fluorescence signals are communicated through the ring-type light guide 31 to a photosensor 32.
The scanner-type fluorescence detection apparatus as shown in FIGS. 5A, 5B, 6A and 6B, can solve the conventional problem. However, if a small-sized and low-output excitation light source is used to furthermore miniaturize the whole apparatus, the fluorescence signal becomes extremely feeble and even if a high-sensitivity photosensor such as a photomultiplier tube is used, insufficient sensitivity may result, because the incidence port of the ring-type light guide (21, 31) used for communicating the fluorescence signal generally is narrow as several hundred xcexcm and the fluorescence signal communication efficiency is low. Particularly, in the scanner-type fluorescence detection apparatus as shown in FIGS. 6A and 6B, the second light guide which rotates is placed in series in addition to the ring-type light guide 31 placed still and a signal is communicated therebetween, thus the scanner-type fluorescence detection apparatus easily falls into insufficient sensitivity to extremely feeble fluorescence. If a high-output excitation light source such as an argon ion laser is used, the problem of insufficient sensitivity is resolved, but a large space is required for the excitation light source together with a control power supply, presenting an obstacle to miniaturization of the apparatus.
Thus, the fluorescence detection apparatus for monitoring a fluorescence signal in real time, particularly for monitoring a fluorescence signal in real time while incubating a sample at a predetermined temperature need satisfy the requirements of (a) high-accuracy temperature adjustment, (b) rapid treatment of a large number of samples, (c) high sensitivity, (d) high reliability (decrease in mechanical trouble typified by broken line, moving part operation failure, etc., improvement in reproducibility of fluorescence detection, decrease in the risk of carry-over), (e) low costs (simplification of apparatus configuration, use of no expensive parts in data processing, etc.,), (f) miniaturization of the apparatus, and the like.
It is therefore an object of the invention to provide a fluorescence detection apparatus satisfying the requirements and in particular to a fluorescence detection apparatus using a small-sized and high-sensitivity optical system of fluorescence analysis, useful for monitoring a large number of fixed samples in real time and a fluorescence detection apparatus provided with an incubation function of temperature control means.
To the end, according to a first aspect of the invention, there is provided a fluorescence detection apparatus comprising a sample holder for fixing and holding sample vessels on a circular arc, a partition plate being joined to drive means for rotation on the center of the circular arc, an excitation light source, excitation light optical means, and fluorescence optical means being fixed on the partition plate for rotation integrally, and a photosensor being mechanically discontinued from the drive means and fixedly placed. In the fluorescence detection apparatus,
(a) the excitation light optical means is placed so as to guide excitation light from the excitation light source from the rotation center side of the partition plate and selectively excite one of the sample vessels,
(b) the fluorescence optical means contains a light guide for communicating a fluorescence signal from the sample vessel to the photosensor and an incidence end of the light guide is placed so as to be able to face the sample vessel with the partition plate between so that the fluorescence signal can be gathered and an emission end of the light guide is placed so as to be able to face the photosensor on the rotation center axis of the partition plate, and
(c) while the excitation light is guided to the sample vessels placed on the circular arc in order as the partition plate is rotated, fluorescence is detected through the fluorescence optical means containing the light guide at the same time.
To the end, according to a second aspect of the present invention, the fluorescence detection apparatus as shown in the first aspect further includes wavelength dispersion means facing the emission end of the light guide for dispersing fluorescence into optical paths depending on the wavelength of the fluorescence signal, wherein a photosensor is fixedly placed on each of the optical paths, whereby fluorescence signals having a plurality of wavelengths can be detected at the same time.
To the end, according to a third aspect of the present invention, the fluorescence detection apparatus as shown in the first or second aspect further includes a shading plate for covering at least the top of one sample vessel, the shading plate being fixed to the partition plate so as to be positioned above the fluorescence incidence end of the light guide forming a part of the fluorescence optical means. Then the shading plate is rotated with the partition plate integrally, thereby shielding at least the sample vessel under fluorescence measurement in order from extraneous light.
To the end, according to a fourth aspect of the present invention, in the fluorescence detection apparatus as shown in the first, second or third aspect, a light emitting diode or a semiconductor laser is used as the excitation light source.
To the end, according to a fifth aspect of the present invention, in the fluorescence detection apparatus as shown in the first, second, or third aspect, the light guide being one component of the fluorescence optical means is one optical fiber.
To the end, according to a sixth aspect of the present invention, in the fluorescence detection apparatus as shown in the second or third aspect, the wavelength dispersion means for dispersing fluorescence into optical paths depending on the wavelength of the fluorescence signal is a dichroic mirror.
To the end, according to a seventh aspect of the present invention, the fluorescence detection apparatus as shown in the first, second, or third aspect further includes temperature adjustment means for controlling each sample at a predetermined temperature.